Daniela D’Arcangelo

Acidosis Inhibits Endothelial Cell Apoptosis and Function and Induces Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor Expression

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

Acidification Prevents Endothelial Cell Apoptosis by Axl Activation

Abstract— Prior studies have shown that acidification due to hypercarbia protects endothelial cells from serum deprivation–induced apoptosis. However, the mechanism(s) responsible for the antiapoptotic effect of acidification is still unclear. cDNA array screening was performed on human umbilical vein endothelial cells cultured in a bicarbonate medium equilibrated either with 5% CO2 (pH 7.4) or with 20% CO2 (pH 7.0). Tyrosine kinase receptor Axl expression was 3.3-fold higher after 6 hours at pH 7.0 compared with pH 7.4; this modulation was confirmed by reverse transcriptase–polymerase chain reaction (3.0±0.9-fold, P <0.03; n=3), Northern blot (3.6±0.1-fold, P <0.0003; n=3), and Western blot (10±1.8-fold, P <0.004; n=3). In a time-course study, both Northern and Western blot analyses showed that the most marked difference in Axl expression between pH 7.4 and pH 7.0 occurred after 24 to 48 hours. Furthermore, Axl phosphorylation was enhanced at pH 7.0. Axl ligand, the survival factor growth arrest–specific gene 6 product (Gas6), was released into the conditioned medium, and by Western blot analysis, similar amounts of protein were found at pH 7.0 and 7.4. Full-length Axl cDNA overexpression reduced serum deprivation–induced apoptosis by 64.4±11.9% in human umbilical vein endothelial cells cultured at pH 7.4 compared with mock-transfected cells (P <0.0004). Furthermore, overexpression of either soluble Axl or antisense Gas6 mRNA partially reverted the protective effect of acidification, increasing ≈2.5-fold the number of apoptotic cells at pH 7.0 (control 19.3±2.7%, soluble Axl 48.9±9.7%, P <0.001; antisense Gas6 49.3±14.3%, P <0.03). In conclusion, Gas6/Axl signaling may play an important role in endothelial cell survival during acidification. The full text of this article is available at http://www.circresaha.org.

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.

The Role of p16INK4a Pathway in Human Epidermal Stem Cell Self-Renewal, Aging and Cancer

The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment. p16INK4a is a potent inhibitor of the G1/S-phase transition of the cell cycle. p16INK4a governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress p16INK4a expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate p16INK4a expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of p16INK4a regulators in human epidermal SC self-renewal, aging and cancer.

Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

Letter to the Editor: “Ion Channels in Brain Metastasis”—Ion Channels in Cancer Set up and Metastatic Progression

The review by Klumpp, L. et al. entitled Ion Channels in Brain Metastasis [1] discusses the role of ion channels in breast cancer, lung cancer and melanoma in metastatic tropism to the brain [...].

Role of “shared epitope” and other citrullination-sites in rheumatoid arthritis and in melanoma.

The manuscript by Koushik S et al. [1] refers to the role of PAD4 enzyme, known to convert arginine to citrulline, as a key posttranslational modification. Citrullination sites are reported to be strongly involved in rheumatoid arthritis (RA) (more than 300 manuscripts in PubMed refer to ‘citrullination’ AND ‘rheumatoid’) as well as in several other disorders such as diabetes, systemic lupus erythematosus, multiple sclerosis, or even interstitial lung disease and other inflammation-based diseases. A given short peptide sequence named ‘shared epitope’ (QKRAA, QRRAA, or RRRAA sequences) is a known citrullination site. It is present within the HLA-DRβ1, is often associated with RA [2,3] and has been reported to physically bind TCR and other proteins [3–5]. Under such regard we would like to underline the potential role of bacteria-infections as possible trigger-mechanisms. For instance, a recent evidence shows that infection by anaerobic Gram-negative bacteria such as Aggregatibacter actinomycetemcomitans (Aa) links periodontal infection to autoimmunity in RA [6] likely as a consequence of hypercitrullination induced in host neutrophils. We would like to underline that different proteins in Aabacterium have these exact epitope sequences with 5/5 amino acid identity: namely RNA chaperone ProQ, ABC transported substratebinding protein, ProP expression regulator, hypothetical protein SA508_10515, and formate dehydrogenase accessory protein FdhE. The short length of such epitopes makes it hard to evaluate the statistical significance of these findings. However, the occurrence of these epitopes suggests that such proteins may potentially trigger an autoimmune reaction and may represent a possible RA trigger mechanism. On the other hand, QKRAA, QRRAA, and RRRAA sequences are present in several proteins of other pathogens. As an example, proteins of Clostridium tetani contain the QKRAA sequence (pyruvate:ferredoxin (flavodoxin) oxidoreductase, molecular chaperone DnaJ, V-type ATP synthase subunit D, terminase, GTP pyrophosphokinase, histidine kinase, and many other proteins), as well as the QRRAA sequence (present in carboxyl-terminal processing protease, stage V sporulation protein B, phosphogluconate dehydrogenase (NADP(+)dependent, decarboxylating, and several more) and the RRRAA sequence (present in transcriptional regulatory protein, sporulation protein YqfD, hydroxymethylbilane synthase, ribonucleoside-diphosphate reductase, and several more). Similarly, such shared epitopes are largely present in proteins of other pathogens such as Brucella, Campylobacter, Enterococcus, Pseudomonas aeruginosa, Salmonella, Streptococcus pneumoniae, and many others pathogens. This finding supports the hypothesis that many pathogens may, at least in part, support potential autoimmune reactions. Koushik et al. [1] also underline a role of citrullination in several other diseases. To this regard, as a further comment, it should be noted that autoantigens modified by citrullination have been recently shown to be produced within an autophagy process and to initiate a CD4-mediated autoimmune response able to exert a potent anti-melanoma action in vivo [7]. Such observation opens an interesting scenario on the possible relation of melanoma progression with pathogens infection as we and others have previously proposed [8–11]. We thus underline here that epitopes-citrullination represent both a RA-triggering autoimmune reaction and a mechanism triggering defensive immune reaction against melanoma epitopes. The ‘shared epitope’ and other sites of citrullination may therefore represent, although under different perspectives, potential therapeutic targets in both RA and melanoma conditions. The pure occurrence of the ‘shared epitope’ does not represent a mechanistic demonstration per se, since the occurrence of such pentapeptides is necessary but not sufficient to recognize and bind target proteins [3]. Nevertheless their presence may underlie, at least in part, the increased melanoma occurrence observed in RA patients under TNF inhibitors treatment [12], although this observation is still debated [13].